The objective of the proposed research is to develop active-site-directed reagents which are irreversible inhibitors of acyl-CoA utilizing enzymes. Specifically, we will attempt to develop reagents which are irreversible inhibitors of acyl-CoA hydrolases, but which will not inhibit acyltransferases. The effect of these reagents on other acyl-CoA utilizing enzymes which play key roles in major metabolic pathways will also be tested. Eventually, we intend to exploit these reagents in studies of lipid metabolism in the brain. The reagents which we propose to synthesize and test are all analogs of CoASH. Highly reactive functional groups, each of which has been shown to react with amino acid side chains by other workers, will be incorporated into CoASH at the thiol end of the molecule. Formation of an enzyme-reagent complex (covalently bonded) will be confirmed by: (a) demonstration of loss of enzyme activity after mixing enzyme and reagent, (b) passing the enzyme and reagent mixture through a Sephadex G-25 column and analyzing the eluted enzyme for the presence of reagent. The various active-site-directed reagents will also be tested as irreversible allosteric activators of pyruvate carboxylase, an enzyme which obligatorily requires the presence of acyl-CoA for activity.